Most epifluorescence microscopy systems use arc lamp or incandescent halogen lamp illuminators to produce light for sample excitation. Arc lamps have the disadvantage of short lifespan and high expense. Certain applications of fluorescence microscopy require rapid on-off switching of the epifluorescence illumination. However, arc lamps cannot be quickly turned on and off, due to significant warm-up and cool-down periods and because of the risk of electrode damage in some types of arc lamps. They are therefore limited in their ability to monitor rapid responses to changes in excitation. Such lamps also take several minutes to produce a stable, constant light output as they warm up.
Some manufacturers have produced fluorescence excitation illuminators that make use of incandescent halogen lamps, but these lamps do not produce ultraviolet light required to excite certain fluorescent dyes. Also, halogen lamps cannot be rapidly turned on and off due to significant warm-up and cool-down periods. Both arc lamps and incandescent lamps require external shutters to rapidly expose the dye to light and cut the exposure off, and various mechanical means are required to direct the excitation light alternately through one wavelength selector device, then another. These mechanical devices cause vibration and timing problems.
Prior art illuminators have some additional limitations and disadvantages. They require high power transformers and draw large amounts of current, which limits their usefulness in portable or mobile applications. For incandescent halogen lamps, UV output is limited. Electromagnetic interference is generated during turn on (ignition) of arc lamps. Prior art arc lamps often produce more light than is necessary, which requires neutral density filters to attenuate unnecessary light output. Light output from arc lamps cannot be modulated, and modulating the output of halogen lamps results in a nonlinear shifting of colour temperature. Large fluxes of heat are produced (25-300 Joule/second) which must be removed, either by means of airflow or waterflow, and airflow cooling can lead to temperature fluctuations at the light source that cause spectral fluctuations in arc lamp or incandescent lamp output. Heat production also means that a heat filter must be used to prevent infrared light from saturating video cameras or other light-detecting elements. Excessive heat production prevents placing heat-sensitive optical elements such as bandpass filters close to the arc lamp or halogen lamp. Sometimes, enough heat is produced to cause significant heating of the rooms in which traditional light sources are located, requiring increased airflow, or addition of air conditioning. There are also safety concerns with arc lamps, including bulb explosion, burns caused contacting hot elements, and ozone production.
Laser light sources have been used for fluorescence microscopy, but the narrow, collimated beam of a laser requires scanning of the light source repetitively across the sample specimen within the field of view of the microscope. There are also speckling artifacts and out-of-plane-of-focus image artifacts associated with laser light sources for fluorescence microscopy. Laser light sources are also relatively expensive.
There is accordingly a need for a light source for exciting fluorescent and phosphorescent molecules via epifluorescence which is less expensive than current fluorescence illuminators, which has lower intrinsic fluctuations (noise), which lasts for a much longer time, and which can be rapidly switched on and off, thereby obviating the need for shutters, optical choppers, optical filter changing devices, and the like. Rapid modulation of intensity would also allow for adaption of intensity for optimizing the fluorescent signal and also permit a number of spectroscopic applications such as fluorescence life-time analysis that currently require more expensive laser light sources, and phosphorescence lifetime analysis that currently requires mechanical shuttering devices.